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Gene Expression Analysis & Genotyping

Techniques geared towards understanding gene regulation have provided higher levels of insight into cellular pathways, differentiation and abnormal or pathological processes. Going beyond classic DNA and RNA quantification , gene expression analysis and genotyping have many applications and are used extensively in medical research. Gene expression analysis is the study of how genes are transcribed into mRNA, then translated into functional gene products including mature RNA, subtypes thereof, and proteins. Genotyping includes analyzing germline DNA to identify the specific nucleotides and bases present. Though seemingly small, these tiny genetic variations can ultimately lead to major changes in the output phenotype.

 

 

Gene Expression Analysis


In research, gene expression analysis can be used to understand biochemical pathways, and aid in expanding and generating databases. Gene expression analysis not only plays a role in disease diagnosis, but has allowed drug discovery and development to expand into tailored therapeutics targeted at specific pathologies.

Gene expression analysis encompasses many techniques, though commonly RNA expression analysis techniques are used because by determining what mRNA transcripts are present within a cell, gene expression can be secondarily concluded. Traditionally, Northern blot has been used to analyze and provide semi-quantitative information about various levels of RNA species in cells and tissues. Additionally, promoter analysis may be performed through ChIP, gel shift assays, or a nuclear run-on assay, that analyzes distinct levels of RNA in a sample.

Instead of RNA analysis, proteins—as they are the functional products of gene expression—can also be analyzed to interpret gene expression through Western blot or other immunoassays. Since gene expression may be impacted by altered chromatin structure and the recruitment of histone modifiers, gene expression analysis can also be performed through post-translational modification analysis via immunoassays and mass spectrometry (MS).

 

Genotyping


Genotyping is commonly used to explore genetic variants, including single nucleotide and copy number variants, though it can also assess large changes in the structural aspects of DNA. It can be used to describe characteristic differences between distinct cellular subtypes, or people, within a population. Genotyping can be used to characterize single alleles, multiple alleles, banding patterns, loci variations, or in virulotyping.

Like gene expression analysis, there are many techniques associated with genotyping depending on the desired target. MALDI couples time of flight (TOF) MS can be used to genotype single nucleotide polymorphisms (SNPs). Popular techniques involving PCR also commonly utilize restriction or amplified fragment length polymorphism (RFLP and AFLP), and random amplified polymorphic DNA (RAPD) techniques that instead target genomic DNA.
 

Methods/Techniques



Used for Both Gene Expression Analysis and Genotyping


Helixyte Green
Quantitative PCR results targeting GAPDH with an input of 100 ng-0.00001 ng cDNA was performed using Helixyte™ Green *20X Aqueous PCR Solution* and a Fast Advanced Master Mix on an Applied Biosystems® 7500 FAST Real-Time PCR System.
Real time qPCR is a technique used in both gene expression analysis and genotyping that offers high levels of accuracy, repeatability, sensitivity, and fast test results. Real time qPCR is based on the principles of using reference genes as endogenous controls to identify abnormalities within the genome. It can be used to measure the expression of specific genes of interest, even at very low expression levels, and can secondarily be used to validate microarray experiments and to monitor biomarkers.

Quantitative real time PCR goes beyond simply detecting DNA, and provides information on how much DNA or gene is present in the sample. Additionally, it can identify SNPs for genotyping applications. Though real time qPCR is widely used in the field of gene expression and genotyping, it typically requires the use of expensive equipment and reagents.


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DNA microarrays can be used for expression analysis of large scale, whole genome experiments, and can monitor the expression levels of thousands of genes simultaneously. DNA microarrays are based on the use of oligonucleotide probes, which are composed of a sequence that is representative of a known RNA species.

Thousands to millions of these probes are fixed to a solid surface, and will attract and hybridize to RNA species in a sample during testing. Analysis can therefore be used to quantitate the relative levels of different RNAs in a sample, and provide genetic information by measuring the frequency of these species through sequencing.

DNA microarrays can be applied in massively parallel technologies, and can help identify SNPs or larger structural changes in a gene of interest. DNA microarrays may be a costlier alternative to other RNA sequencing techniques, and as DNA microarray databases are used for interpreting data, analysis is reliant upon the existing knowledge of the gene sequence.
 

Used for Gene Expression Analysis


RNA-seq workflow

Simplified workflow of RNA-Seq, from isolated RNA (fragmented as necessary) to post-sequencing expression level estimations. Infographic built using Biorender.
 
RNA-Seq is a method based on high-throughput next generation sequencing (NGS) technologies. In RNA-Seq, sequences of base pairs, also termed reads, are first sequenced then aligned with a reference genome. These reads can be assembled into transcripts, then the expression level of each gene is estimated by counting the number of reads that align into the transcript.

This method can provide a high-resolution view of the transcriptome, as well as an incredibly detailed and quantitative view of gene expression. RNA-Seq can also give information regarding alternative splicing and allele-specific expression, and has been used to identify new biomarkers, novel gene structures, or genetic variants, and mutations. Unfortunately, the procedure may be costly, and analysis can be time consuming. Careful consideration must also be made at each step in the experimental process to prevent any source of potential bias before analysis.

RNase protection assays (RPA) have also been used to detect, quantify and characterize specific mRNA species with sensitivity and specificity. In RPA, labeled antisense RNA probes hybridized to complementary target sequences in a sample. After, all remaining non-target single strand RNAs are digested, and standard curves can be generated to reveal the changes and levels of mRNA in tissues.
StrandBrite vs. SYTO
RNase and DNase digest test of HeLa cells stained with (A) StrandBrite™ Green RNA or (B) SYTO RNASelect, respectively. Both RNA probes were tested in the concentration of 1.5 µM. DNase of 50 U/mL and RNase of 50 µg/mL were added into cells. Fluorescence images were taken using a fluorescence microscopy with FITC filter.


As RPA utilizes gel electrophoresis for analysis, this method offers a cost-effective alternative to other RNA expression analysis methods. Some disadvantages to RPA lie in that products are limited to the size of initial probes used, and the natural occurrence of posttranslational processing may mean that mRNA levels in a sample are not always directly indicative of the actual levels of gene expression.

Used for Genotyping


DNA-Seq is another technique that can be used for genotyping applications. Like RNA-Seq, this method also incorporates the use of NGS, and can determine the exact order of nucleotides, and conclude the exact sequence, of a specific DNA molecule.

This process works by simultaneously identifying DNA bases while incorporating them into a growing nucleic acid chain. Each base will then emit a unique fluorescent signal when it pairs to the strand, and analysis includes the identification and quantification of these signals. DNA-Seq offers high throughput capabilities, is extremely sensitive and has a dynamic range of ability from a small target region or even an entire genome. The nature of DNA-Seq allows researchers the ability to get pictures on a base-by-base case, solely for the exome, or even for the full genome. The biggest disadvantage of this method is that vast amounts of data are provided, which may make analysis difficult, time-consuming, and increase overall costs.
 

Product Ordering Information


 

Table 2. RNA quantification and PCR reagents

Product Name
Ex (nm)
Em (nm)
Unit Size
Cat No.
StrandBrite™ Green Fluorimetric RNA Quantitation Kit *Optimized for Microplate Readers*490 nm545 nm1000 Tests17655
StrandBrite™ Green Fluorimetric RNA Quantitation Kit490 nm540 nm100 Tests17656
StrandBrite™ Green Fluorimetric RNA Quantitation Kit *High Selectivity*490 nm540 nm100 Tests17657
StrandBrite™ Green RNA Quantifying Reagent490 nm525 nm1 mL17610
StrandBrite™ Green RNA Quantifying Reagent490 nm525 nm10 mL17611
Portelite™ Fluorimetric RNA Quantitation Kit490 nm525 nm100 Tests17658
Portelite™ Fluorimetric RNA Quantitation Kit490 nm525 nm500 Tests17659
Cyber Green™ [Equivalent to SYBR® Green] *20X Aqueous PCR Solution*498 nm522 nm5 x 1 mL Tests17591
Cyber Green™ [Equivalent to SYBR® Green] *20X Aqueous PCR Solution*498 nm522 nm1 mL17592
Cyber Green™ Nucleic Acid Gel Stain [Equivalent to SYBR® Green]498 nm522 nm100 µL17604
Cyber Green™ Nucleic Acid Gel Stain [Equivalent to SYBR® Green]498 nm522 nm1 mL17590

Table 3. TAQuest™ qPCR Master Mixes.

Product
Reference Dye
Unit Size
Cat No.
TAQuest™ qPCR Master Mix with Helixyte™ Green No Rox 1 mL 17270
TAQuest™ qPCR Master Mix with Helixyte™ Green No Rox 5 mL 17271
TAQuest™ qPCR Master Mix with Helixyte™ Green Low Rox 1 mL 17272
TAQuest™ qPCR Master Mix with Helixyte™ Green Low Rox 5 mL 17273
TAQuest™ qPCR Master Mix with Helixyte™ Green High Rox 1 mL 17274
TAQuest™ qPCR Master Mix with Helixyte™ Green High Rox 5 mL 17275
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green No Rox 1 mL 17276
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green No Rox 5 mL 17277
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green Low Rox 1 mL 17278
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green Low Rox 5 mL 17279
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green High Rox 1 mL 17280
TAQuest™ FAST qPCR Master Mix with Helixyte™ Green High Rox 5 mL 17281
TAQuest™ qPCR Master Mix for TaqMan Probes No Rox 1 mL 17282
TAQuest™ qPCR Master Mix for TaqMan Probes No Rox 5 mL 17283
TAQuest™ qPCR Master Mix for TaqMan Probes Low Rox 1 mL 17284
TAQuest™ qPCR Master Mix for TaqMan Probes Low Rox 5 mL 17285
TAQuest™ qPCR Master Mix for TaqMan Probes High Rox 1 mL 17286
TAQuest™ qPCR Master Mix for TaqMan Probes High Rox 5 mL 17287
TAQuest™ FAST qPCR Master Mix for TaqMan Probes No Rox 1 mL 17288
TAQuest™ FAST qPCR Master Mix for TaqMan Probes No Rox 5 mL 17289
TAQuest™ FAST qPCR Master Mix for TaqMan Probes Low Rox 1 mL 17290
TAQuest™ FAST qPCR Master Mix for TaqMan Probes Low Rox 5 mL 17291
TAQuest™ FAST qPCR Master Mix for TaqMan Probes High Rox 1 mL 17292
TAQuest™ FAST qPCR Master Mix for TaqMan Probes High Rox 5 mL 17293

 

Further Reading


The RNase Protection Assay
RNase Protection Assay
Gene Expression Analyzed by Ribonuclease Protection Assay
Revisiting Global Gene Expression Analysis
RNA Sequencing and Analysis
Comparison of three PCR-based assays for SNP genotyping in plants
Genotyping Technologies for Genetic Research
Research Techniques Made Simple: Polymerase Chain Reaction (PCR)